ES2562256T5 - VWF purification to increase clearance of non-enveloped virus - Google Patents

Description

DESCRIPTION

VWF purification to increase clearance of non-enveloped virus

This application claims the benefit of priority under 35 U.S.C. § 119(e) of US Provisional Application No. 61/235,570, filed August 20, 2009.

FIELD OF THE INVENTION

In general, the invention relates to methods of purifying VWF to enhance clearance of non-lipid enveloped virus.

BACKGROUND OF THE INVENTION

Von Willebrand factor (VWF) is a circulating plasma glycoprotein as a series of multimers ranging in size from approximately 500 to 20,000 kD. Multimeric forms of VWF are composed of 250 kD polypeptide subunits linked together by disulfide bonds. VWF is involved in initial platelet adhesion to the subendothelium of the damaged vascular wall. Only the largest multimers exhibit hemostatic activity. It is assumed that endothelial cells secrete large polymeric forms of VWF and that low molecular weight forms of VWF (low molecular weight VWF) are the result of proteolytic cleavage. High molecular weight multimers are stored in the Weibel-Pallade bodies of endothelial cells and are released upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as prepro-VWF, which consists largely of repeat domains. After dissociation of the signal peptide, pro-VWF dimerizes through disulfide bonds in its C-terminal region. The dimers serve as promoters for multimerization, which is controlled by disulfide bonds between the free termini. After assembly into multimers, proteolytic removal of the propeptide sequence occurs (Leyte et al., Biochem. J. 274 (1991), 257-261).

The predicted primary translation product of the cloned VWF cDNA is a precursor polypeptide (prepro-VWF) of 2,813 residues. Prepro-VWF consists of a 22 amino acid signal peptide and a 741 amino acid propeptide, with mature VWF comprising 2050 amino acids (Ruggeri Z.A., and Ware, J., FASEB J., 308-316 (1993)).

Defects in VWF are the cause of von Willebrand disease (VWD), which is characterized by a more or less pronounced hemorrhagic phenotype. VWD type 3 is the most severe form, where VWF is completely absent, and VWD type 1 refers to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 refers to qualitative VWF defects and can be as severe as VWD type 3. VWD type 2 has many subforms, some of which are associated with the loss or decrease of high molecular weight multimers . Von Willebrand syndrome type 2a (VWS-2A) is characterized by a loss of both intermediate and large multimers. SVW-2B is characterized by a loss of the higher molecular weight multimers. Other diseases and conditions related to VWF are known in the art.

The removal or inactivation of non-enveloped viruses from therapeutic protein solutions is traditionally accomplished with treatments by physical methods, such as high temperature (eg, dry heat, steam heat, pasteurization), irradiation with high-energy rays (eg, ultraviolet (UV) or beta radiation), low pH, nanofiltration, or by chromatographic methods, in particular affinity chromatography. However, these treatments are often ineffective in purifying a high molecular weight protein such as VWF, which does not pass through a nanofilter and/or loses its potency or molecular integrity when treated with heat or radiation.

Cameron R et al., Biologicals (1997), vol. 25, no. 4, pages 391-401, refers to the purification of human albumin using ion exchange chromatographic procedures. Bollag DM et al., 1996, Wiley-Liss, Inc., New York, NY, "Protein Methods (2nd Edition)" Chapter 9 describes methods of purifying proteins using ion exchange chromatography. US 6,465,624 B1 relates to a method for recovering von Willebrand factor (vWF) purified by cation exchange chromatography.

Current regulatory guidelines call for manufacturers to address the issue of depletion and/or inactivation of both lipid-enveloped and non-lipid-enveloped viruses for recombinant pharmaceuticals. The ICH "Guideline on Viral Safety Evaluations of Biotechnology Products" (Federal Register, 1998, 63(185): 51074-51084) gives manufacturers flexibility on how to address virus issues taking into account the type of product, the process of production and the risk of viruses potentially polluting. These guidelines note that the risk of viral contamination is a common feature of all biotech products derived from cell lines. This contamination could have serious clinical consequences and may result from contamination of the source cell lines themselves (cell substrates) or from the accidental introduction of virus during production.

While inactivation of lipid-enveloped viruses can be carried out very effectively following a solvent/detergent (S/D) treatment method, inactivation of model non-lipid enveloped viruses (NLEV) can be difficult due to its small size and physical stability.

Accordingly, there is a need in the art to develop methods for efficiently inactivating or removing non-enveloped virus during VWF purification.

SUMMARY OF THE INVENTION

The present invention provides an effective method of VWF purification to enhance the clearance of non-lipid enveloped virus. The present invention provides a new VWF purification method to enhance NLEV removal by performing the product loading step and the washing step of the purification process at a high pH.

One method known in the art for purifying NLEV polypeptides involves the use of nanofiltration. The principle behind the efficient separation of protein and virus using nanofiltration takes advantage of the difference in size between the polypeptide and the virus; efficient separation requires the polypeptide to have an effective size smaller than that of the virus, allowing the polypeptide to pass through the pores of the nanofilter while the virus is retained. However, if the polypeptide and the virus are of comparable sizes to one another, separation is problematic as either the polypeptide and the virus both pass through the pores of the nanofilter, or neither do. The methods disclosed herein overcome this problem by using a cation exchange resin instead of nanofiltration, and loading and/or washing the resin at a high enough pH to separate the polypeptide from the virus.

Without being limited by theory, the methods described herein are useful for increasing the removal of NLEV from polypeptide solutions where the polypeptide has a certain size and/or conformation. A polypeptide of sufficiently large size is likely to have localized charge characteristics at or above the isoelectric point of the polypeptide, that is, certain regions of the polypeptide may hold localized positive or negative charges, thus allowing the polypeptide to adsorb onto the polypeptide. the resin from the column while the virus flows through it. This irregular charge distribution throughout a polypeptide allows the polypeptide to remain bound to the resin despite loading and/or washing the resin at high pH.

The invention provides a method as defined in the claims.

A method of removing a non-enveloped virus from a protein-containing solution as defined in the claims is provided, comprising the solution on a cation exchange resin at a pH greater than 3.0 units or more than the isoelectric point of the protein and washing the cation exchange resin with a washing buffer to obtain an eluate, said washing buffer having a pH lower than that of the solution applied to the cation exchange resin.

The pH of the solution is at 3 or about 3.1, or about 3.2, or about 3.3, or about 3.4, or about 3.5, or about 3.6, or about 3.7, or about 3.8, or about 3.9, or about 4.0, or about 4.1, or about 4.2, or about 4.3, or about 4.4, or about 4.5, or about 4.6, or about 4.7, or about 4.8, or about 4.9, or about 5.0, or about 5.1, or about 5.2, or about 5.3, or about 5, 4, or about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0 or more pH units, or more above the isoelectric point of the protein. In these embodiments, the pH is greater than about 7. In a related aspect, the pH of the protein-containing solution is about 7.0. In other aspects, the pH of the protein-containing solution is about 7.1, or about 7.2, or about 7.3, or about 7.4, or about 7.5, or about 7.6, or about 7.7, or about 7.8, or about 7.9, or about 8.0, or about 8.1, or about 8.2, or about 8.3, or about 8.4, or about 8 .5, or about 8.6, or about 8.7, or about 8.8, or about 8.9, or about 9.0, or about 9.1, or about 9.2, or about 9.3 , or about 9.4, or about 9.5, or about 9.6, or about 9.7, or about 9.8, or about 9.9, or about 10.0, or about 10.1, or about 10.2, or about 10.3, or about 10.4, or about 10.5, or about 10.6, or about 10.7, or about 10.8, or about 10.9, or about 11 .0, or about 11.1, or about 11.2, or about 11.3, or about 11.4, or about 11.5, or about 11.6, or about 11.7, or about 11.8, or about 11.9, or about 12.0, or about 12 .1, or about 12.2, or about 12.3, or about 12.4, or about 12.5, or about 12.6, or about 12.7, or about 12.8, or about 12.9 , or about 13.0 or higher.

In one embodiment, the solution protein is a polypeptide having a molecular mass of at least about 175 kilodaltons, or about 180 kilodaltons, or about 190 kilodaltons, or about 200 kilodaltons, or about 210 kilodaltons, or about 220 kilodaltons, or or about 230 kilodaltons, or about 240 kilodaltons, or about 250 kilodaltons, or about 260 kilodaltons, or about 270 kilodaltons, or about 280 kilodaltons, or about 290 kilodaltons, or about 300 kilodaltons, or about 310 kilodaltons, or about 320 kilodaltons, or approximately 330 kilodaltons, or approximately 340 kilodaltons, or approximately 350 kilodaltons, or approximately 360 kilodaltons, or approximately 370 kilodaltons, or approximately 380 kilodaltons, or approximately 390 kilodaltons, or approximately 400 kilodaltons, or approximately 410 kilodaltons, or approximately 420 kilodaltons, or about 430 kilodaltons, or about 440 kilodaltons, or about 450 kilodaltons, or about 460 kilodaltons, or about 470 kilodaltons, or about 480 kilodaltons, or about 490 kilodaltons, or about 500 kilodaltons or more. As described herein, polypeptides also comprise multimeric structures, and such multimeric structures, in various aspects, have a molecular mass of at least about 500 kilodaltons. In related aspects, the multimeric structures have a molecular mass of at least about 510, or about 520, or about 530, or about 540, or about 550, or about 560, or about 570, or about 580, or about 590, or about 600, or about 610, or about 620, or about 630, or about 640, or about 650, or about 660, or about 670, or about 680, or about 690, or about 700, or about 710, or about 720 , or about 730, or about 740, or about 750, or about 760, or about 770, or about 780, or about 790, or about 800, or about 810, or about 820, or about 830, or about 840, or about 850, or about 860, or about 870, or about 880, or about 890, or about 900, or about 910, or about 920, or about 930, or about 940, or about 950, or about 960, or about 970 , or about 980, or about 990 kilodaltons, or about 1 megadalton, or about 1.1 megadaltons, or about 1.2 megadaltons, or about 1.3 megadaltons, or about 1.4 megadaltons, or about 1.5 megadaltons, or about 1.6 megadaltons, or about 1.7 megadaltons, or about 1.8 megadaltons, or about 1.9 megadaltons, or about 2.0 megadaltons, or about 2.1 megadaltons, or about 2.2 megadaltons, or approximately 2.3 megadaltons, or approximately 2.4 megadaltons, or approximately 2.5 megadaltons, or approximately 2.6 megadaltons, or approximately 2.7 megadaltons, or approximately 2.8 megadaltons, or approximately 2.9 megadaltons, or approximately 3.0 megadaltons, or about 3.1 megadaltons, or about 3.2 megadaltons, or about 3.3 megadaltons, or about 3.4 megadaltons, or about 3.5 megadaltons, or about 3.6 megadaltons, or about 3 .7 megadaltons, or about 3.8 megadaltons, or about 3.9 megadaltons, or about 4.0 megadaltons, or about 4.1 megadaltons, or about 4.2 megadaltons, or about 4.3 megadaltons, or about 4, 4 megadaltons, or about 4.5 megadaltons, or about 4.6 megadaltons, or about 4.7 megadaltons, or about 4.8 megadaltons, or about 4.9 megadaltons, or about 5.0 megadaltons or more.

In some embodiments, the cation exchange resin has a negatively charged group selected from the group consisting of carboxymethyl (CM), sulfoalkyl (SP, SE), sulfate, and methylsulfonate (S), as well as any other negatively charged ligand.

In another embodiment, the protein is a blood coagulation protein. In various aspects, the blood coagulation protein is selected from the group consisting of factor VIII, von Willebrand factor, FI (fibrinogen), FV (proacelerin), FXI (plasma thromboplastin history), and FXIII (fibrin stabilizing factor). .

In one embodiment there is provided a method of removing non-enveloped virus from a solution containing von Willebrand factor (VWF) as defined in the claims, comprising applying the solution to a cation exchange resin at a pH greater than 3 units or more to the isoelectric point of VWF and washing the cation exchange resin with a first wash buffer to form an eluate, said first wash buffer having a pH that is lower than that of the solution applied to the cation exchange resin.

BRIEF DESCRIPTION OF THE FIGURES

In another embodiment, a method of removing a non-enveloped virus from a solution containing VWF is provided, comprising applying the solution to a cation exchange resin with a pH 3 units or more above the isoelectric point of VWF and washing the exchange resin. cationic with a first wash buffer having a pH greater than the isoelectric point of the protein applied to the cation exchange resin; and washing the cation exchange resin with a second wash buffer to form an eluate, said first eluate having a lower pH than the first wash buffer.

Figure 1: Shows the result of an SDS-PAGE separation followed by silver staining (A) and Western Blot analysis (B) for residual rFVIII.

Figure 2: shows the stained gel of UNO S passages with samples with added MMV and REO virus. Figure 3: shows the stained gel of UNO S passages with samples with added MMV and REO virus. Figure 4 shows the results obtained by subjecting the purified rVWF preparations obtained by the process variants to proteolytic digestion by native V8 protease and separating the resulting peptides by RP-HPLC.

Figure 5: shows the results obtained by subjecting the purified rVWF preparations obtained by the process variants to trypsin in the denatured state and separating the resulting peptides by RP-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a VWF purification method with increased clearance of non-lipid enveloped virus. The methods of the invention are applicable in both column (ie chromatography) and batch mode (ie without column equipment).

The method of the present invention employs a purification method on a cation exchange resin to enhance clearance of non-lipid enveloped virus. Previous VWF purification methods using cation exchange chromatography were performed at neutral pH. These methods made it possible to produce purified VWF in good yield and purity, but surprisingly the process had no ability to remove non-enveloped virus.

Definition of terms

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by a person skilled in the art to which this invention pertains. The following references provide those skilled in the art with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed.

1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5th ed., R. Rieger et al. (eds.), Springer Publishing (1991); and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

Note that, as used in this specification and the appended claims, the singular forms "a", "an", "the" and "the" include plural reference unless the context clearly indicates otherwise. contrary.

As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

As used herein, the terms "express", "expressing" and "expression" mean allowing or causing the information contained in a gene or DNA sequence to be revealed, for example by producing a protein by activating the cellular functions involved in the transcription and translation of a gene or corresponding DNA sequence. A DNA sequence is expressed in a cell or is expressed by a cell to form an "expression product", such as a protein. The expression product itself, eg the resulting protein, can also be said to be "expressed". An expression product can be characterized as intracellular, extracellular, or secreted. The term "intracellular" means within a cell. The term "extracellular" means outside of a cell, for example certain types of transmembrane proteins. A substance is "secreted" by a cell if it appears to a significant extent outside the cell, from somewhere on the cell, or within the cell.

As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues, structural variants, related natural structural variants, and unnatural synthetic analogs thereof linked by peptide bonds. Synthetic polypeptides can be prepared, for example, using an automated polypeptide synthesizer. The term "protein" usually refers to large polypeptides. The term "peptide" usually refers to short polypeptides. The term "polypeptide" also includes polymeric structures. Thus, a polypeptide can be a monomer, a dimer, a trimer or a larger multimeric structure. These multimeric structures can have a molecular mass of up to 5 megadaltons or more.

As used herein, the "isoelectric point" is the pH value at which the net electrical charge of a polypeptide in an aqueous solution is zero.

As used herein, a "fragment" of a polypeptide refers to any part of a polypeptide or protein smaller than the expression product of the full-length polypeptide or protein.

As used herein, an "analog" refers to any two or more polypeptides that are essentially similar in structure and have the same biological activity, but that may have varying degrees of activity relative to the whole molecule or to a fragment. Of the same. The analogs differ in the composition of their amino acid sequences based on one or more mutations that involve the substitution of one or more amino acids with other amino acids. Substitutions can be conservative or non-conservative, depending on the degree of physicochemical or functional resemblance of the substituted amino acid to the amino acid that replaces it.

As used herein, a "variant" refers to a polypeptide, protein, or analog thereof that is modified to include additional chemical moieties that are not normally part of the molecule. These fractions can modulate the solubility, absorption, biological half-life, etc. of the molecule. Alternatively, the fractions can reduce the toxicity of the molecule and eliminate or mitigate undesirable side effects of the molecule, etc. Fractions that can produce these effects are described in Remington's Pharmaceutical Sciences (1980). Procedures for coupling these moieties to a molecule are well known in the art. For example, the variant may be a blood coagulation factor that has a chemical modification that confers a longer in vivo half-life to the protein. In various aspects, polypeptides are modified by glycosylation, pegylation, and/or polysialylation.

recombinant VWF

The polynucleotide and amino acid sequences of prepro-VWF are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and are available from GenBank under accession numbers NM_000552 and NP_000543, respectively. The amino acid sequence corresponding to the mature VWF protein is shown in SEQ ID NO: 3 (corresponding to amino acids 764-2813 of the full length prepro-VWF amino acid sequence).

A useful form of rVWF has at least the property of stabilizing in vivo, eg binding, at least one factor VIII (FVIII) molecule, and optionally having a pharmacologically acceptable glycosylation pattern. Specific examples include VWF without the A2 domain and therefore resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77: 1008-1013, 1997), and the VWF fragment from Val 449 to Asn 730 that includes the binding domain. of glycoprotein Ib and binding sites for collagen and heparin (Pietu et al., Biochem. Biophys. Res. Commun. 164: 1339-1347, 1989). Determination of the ability of a VWF to stabilize at least one FVIII molecule can be carried out in VWF-deficient mammals according to methods known in the art.

The rVWF of the present invention can be produced by any method known in the art. A specific example is described in WO86/06096, published October 23, 1986, and in US Patent Application No. 07/559,509, filed July 23, 1990. Accordingly, methods are known in the prior art for (i) producing recombinant DNA by genetic engineering, for example by reverse transcription of RNA and/or DNA amplification, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, by e.g. electroporation or microinjection, (iii) culturing said transformed cells, e.g. continuously or in batches, (iv) expressing VWF, e.g. constitutively or upon induction, and (v) isolating said VWF, e.g. from culture medium. culturing or harvesting the transformed cells, to (vi) obtain purified rVWF, for example by anion exchange chromatography or affinity chromatography. A recombinant VWF can be produced in transformed host cells using recombinant DNA techniques well known in the art. For example, sequences coding for the polypeptide could be removed from the DNA using suitable restriction enzymes.

Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. It would also be possible to use a combination of these techniques.

The invention also provides vectors that encode polypeptides of the invention in an appropriate host. The vector comprises the polynucleotide encoding the polypeptide operably linked with expression control sequences. Methods for performing this functional linkage, either before or after insertion of the polynucleotide into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, termination signals, polyadenylation signals, and other signals involved in the control of transcription or translation. The resulting vector that includes the polynucleotide is used to transform an appropriate host. This transformation can be carried out using methods known in the art.

Any of a large number of known and available host cells can be used in the practice of this invention. Selection of a particular host depends on a number of art-recognized factors, including, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of peptides, expression characteristics, biosafety and costs. It is necessary to find a balance between these factors knowing that not all host cells are equally efficient for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells include bacteria, yeast and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

The transformed host is then cultured and purified. Host cells can be grown under standard fermentation conditions so that the desired compounds are expressed. These fermentation conditions are known in the art. Finally, the polypeptides from the culture are purified by methods known in the art.

Depending on the host cell used to express a compound of the invention, it may be convenient to attach carbohydrate (oligosaccharide) groups to sites known to be glycosylation sites in proteins. In general, O-linked oligosaccharides bind to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides bind to asparagine (Asn) residues when they are part of the Asn-X-Ser/Thr sequence, where X can be any amino acid except proline. Preferably, X is one of the 19 natural amino acids excluding proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar commonly found in both is N-actylneuraminic acid (called sialic acid). Sialic acid is normally the residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can impart acidic properties to the glycosylated compound. This site(s) can be incorporated into the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (eg in mammalian cells such as CHO, BHK, COS). However, these sites can be further glycosylated by synthetic or semi-synthetic procedures known in the art.

Alternatively, the compounds can be prepared by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable methods are known in the art, including those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; ^US Patent No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid-phase synthesis is the preferred technique for preparing individual peptides, as it is the most cost-effective method for producing small peptides.

VWF fragments, variants and analogues

Methods for preparing polypeptide fragments, variants or analogs are well known in the art.

Fragments of a polypeptide are prepared using, without limitation, enzymatic cleavage (eg trypsin, chymotrypsin) and also using recombinant means to generate a polypeptide fragment with a specific amino acid sequence. It is possible to generate polypeptide fragments that include a region of the protein with a particular activity, such as a multimerization domain or any other identifiable VWF domain known in the art.

Variants of a polypeptide are envisioned herein including human and non-human forms of VWF (eg murine VWF). The methods described herein also envision the use of chimeric polypeptides, for example a mouse/human fusion polypeptide.

Methods for preparing polypeptide analogues are also known. Amino acid sequence analogs of a polypeptide can be substitution, insertion, addition, or deletion analogs. Deletion analogs, including fragments of a polypeptide, are missing one or more residues of the native protein that are not essential for immunogenic function or activity. Insertion analogs involve the addition, for example of amino acid(s), at a non-terminal point of the polypeptide. This analog may include the insertion of an immunoreactive epitope or just a single residue. Addition analogues, including fragments of a polypeptide, include the addition of one or more amino acids at either end of a protein and include, for example, fusion proteins.

Substitution analogs typically exchange one wild-type amino acid for another at one or more sites within the protein, and can be designed to modulate one or more properties of the polypeptide without losing other functions or properties. In one aspect, the substitutions are conservative substitutions. The term "conservative amino acid substitution" means the substitution of an amino acid for an amino acid having a side chain with a similar chemical character. Similar amino acids to make conservative substitutions include those with an acidic side chain (glutamic acid, aspartic acid); a basic side chain (arginine, lysine, histidine); a polar amide side chain (glutamine, asparagine); an aliphatic hydrophobic side chain (leucine, isoleucine, valine, alanine, glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine); a small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine).

Analogs can be essentially homologous or essentially identical to the recombinant VWF from which they are derived. Some preferred analogs are those that retain at least some of the biological activity of the wild-type polypeptide, for example blood clotting activity.

Envisaged polypeptide variants include polypeptides chemically modified by techniques such as ubiquitination, glycosylation, including polysialation, conjugation with therapeutic or diagnostic agents, labeling, covalent polymeric attachment such as pegylation (polyethylene glycol derivatization), introduction of non-hydrolyzable bonds, and insertion or substitution. by chemical synthesis of amino acids such as ornithine, which are not normally present in human proteins. The variants retain the same or essentially the same binding properties as the unmodified molecules of the invention. This chemical modification may include direct or indirect attachment (eg, through a linker) of an agent to the fVw polypeptide. In the case of an indirect connection, the possibility that the linker is hydrolyzable or non-hydrolyzable is provided.

In general, the preparation of pegylated polypeptide analogs will comprise the steps of (a) reacting the polypeptide with polyethylene glycol (as a reactive steric or aldehydic derivative of PEG) under conditions in which the ligation construct polypeptide binds to one or more PEG groups, and (b) obtaining the reaction product(s). In general, the optimal reaction conditions for acylation reactions will be determined based on known parameters and the desired result. For example, the higher the PEG:protein ratio, the higher the percentage of polypegylated product. In some embodiments, the ligation construct will have a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) may be added to the blood coagulation factor to achieve a longer in vivo half-life. The PEG group can have any convenient molecular weight and can be linear or branched. The average molecular weight of PEG ranges from about 2 kilodaltons ("kDa") to about 100 kDa, from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10 kDa. PEG groups are attached to blood coagulation factor by reductive acylation or alkylation via a natural or artificial reactive group on the PEG moiety (for example, an aldehyde, amino, thiol, or ester group) to a reactive group on the coagulation factor blood (for example an aldehyde, amino or ester group) or by any other known technique.

In US Patent Publication 20060160948, Fernandes and Gregoriadis; Biochim. Biophys. Acta 1341:26-34, 1997, and Saenko et al., Haemophilia 12:42-51, 2006, describe methods for preparing polysialylated polypeptide. Briefly, a solution of colominic acid containing 0.1M NaIO ₄ is stirred in the dark at room temperature to oxidize the AC. The activated CA solution is dialyzed, for example against 0.05M sodium phosphate buffer, pH 7.2, in the dark, and this solution is added to rVWF solution and incubated for 18 hours at room temperature in the dark under gentle stirring. Free reagents can then be separated from the rVWF-polysialic acid conjugate by ultrafiltration/diafiltration. Conjugation of rVWF with polysialic acid can also be achieved using glutaraldehyde as a crosslinking reagent (Migneault et al., Biotechniques 37:790-796, 2004).

It is also envisioned that a polypeptide of the invention may be a fusion protein with a second agent that is a polypeptide. In one embodiment, the second agent that is a polypeptide is, without limitation, an enzyme, a growth factor, an antibody, a cytokine, a chemokine, a cell surface receptor, the extracellular domain of a cell surface receptor, a cell adhesion molecule, or a fragment or an active domain of a protein described above. In a related embodiment, the second agent is a blood coagulation factor, such as factor VIII, factor VII, factor IX. The intended fusion protein is prepared by chemical or recombinant techniques known in the art.

It is also anticipated that prepro-VWF and pro-VWF polypeptides may provide therapeutic benefit in the formulations of the present invention. For example, US Patent No. 7,005,502 describes a pharmaceutical preparation comprising essential amounts of pro-VWF that induce thrombin generation in the presence of platelets in vitro. In addition to biologically active recombinant fragments, variants or analogs of natural VWF, the present invention envisions the use of fragments, variants or analogs biologically active recombinants of prepro-VWF polypeptides (shown in SEQ ID NO: 2) or pro-VWF polypeptides (amino acid residues 23 to 764 of SEQ ID NO: 2) in the formulations described herein.

Polynucleotides encoding fragments, variants, and analogs can be readily generated by a skilled worker to encode biologically active fragments, variants, or analogs of the native molecule that have the same or similar biological activity as the native molecule. These polynucleotides can be prepared using PCR techniques, digestion/ligation of coding DNA molecules, and the like. Thus, one of ordinary skill in the art will be able to generate single base changes in the DNA strand to obtain a codon missense and reverse missense mutation, using any method known in the art, including, but not limited to, site-specific mutagenesis. . As used herein, the phrase "moderately stringent hybridization conditions" means, for example, hybridization at 42°C in 50% formamide and washing at 60°C in 0.1 x SSC, 0.1% SDS. Those skilled in the art will understand that a variation of these conditions occurs depending on the length and GC nucleotide base content of the sequences to be hybridized. Standard formulas in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Methods to produce VWF

Industrially, mVWF, in particular human recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in a genetically engineered CHO cell line. The function of co-expressed rVWF is to stabilize rFVIII in the cell culture process. rVWF is synthesized in the cell as the "pro" form, which contains a large propeptide attached to the N-terminus. After its maturation in the endoplasmic reticulum and Golgi apparatus, the propeptide is cleaved by the action of the cellular furin protease and secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein.

VWF purification

Provided herein is a method of removing non-enveloped virus from a protein-containing solution as defined in the claims, comprising applying the solution to a cation exchange resin with a pH of 3 units or more above the isoelectric point of the protein, and washing the cation exchange resin with a wash buffer to form an eluate, said wash buffer having a pH lower than that of the solution applied to the cation exchange resin.

The pH of the solution is 3.0 or about 3.2 or about 3.4, or about 3.6, or about 3.8, or about 4.0, or about 4.2, or about 4.4, or about 4.6, or about 4.8, or about 5.0, or about 5.5, or about 6.0 units or more above the isoelectric point of the protein.

The following examples are not intended as limiting examples, but merely illustrate specific embodiments of the invention.

examples

Example 1

The viruses and cells used in the assays described below are the following:

REO-3 (Family Reoviridae; non-enveloped dsRNA virus), Strain Dearing (ATCC VR-824) was obtained from ATCC. Virus was propagated and titrated in Vero cells obtained from ECACC (84113001). Mm V (Family Parvoviridae; non-enveloped ssDNA virus), the prototype strain (ATCC VR-1346), was obtained from the American Type Culture Collection, Rockville, Maryland. The virus was propagated and titrated in A9 cells (ATCC CCL-1 .4). PPV (Family Parvoviridae; non-enveloped ssDNA virus), Tennessee strain (BRFF #PP951024), was obtained from Biological Research Faculty & Facility, Ijamsville, Maryland. The virus was propagated and titrated in PK-13 cells (ATCC CRL-6489). EMCV (Family Picornaviridae; non-enveloped ssRNA) (ATCC #VR-129B) was obtained from the American Type Culture Collection. The virus was propagated and titrated in Vero cells (European Collection of Cell Cultures, E ^c A ^c C, #84113001). HadV (Family Adenoviridae; non-enveloped dsDNA), strain Adenoid 75 (ATCC VR-5), was obtained from the American Type Culture Collection. The virus was propagated and titrated in HeLa cells (ATCC CCL-2).

Steps included in an example VWF purification process include:

• Immunoaffinity chromatography of cell culture supernatant.

Yo. Circulation fraction.

• Anion exchange (eg trimethylaminoethyl anion exchange column).

• Filtration (0.45/0.2 μm).

• Anion exchange (eg Mustang Q (Pall Corporation)).

• Viral inactivation (eg by solvent/detergent treatment).

• Filtration (0.8/0.65 |jm).

• Cation exchange (eg UNO S column).

• Ultrafiltration/concentration.

• Filtration (0.45/0.2 jm ).

• Gel filtration (Superose 6, prep grade (GE Life Sciences)).

Optimization of the passage of UNO S . During the UNO S step, the rVWF binds to a strong cation exchange resin while some of the impurities pass through the column. After washing the column with high conductivity buffers, bound rVWF is released from the column with a salt step. During initial viral clearance studies, this step showed at least a significant clearance rate of the REO virus model. The conditions of the applied parameters and the corresponding results are shown in the following Table 1.

Table 1

As can be seen in Table 1, moderate changes in process parameters (conductivity modification, pH 8.0, and additives to wash buffers) did not lead to any significant improvement in mMv removal rates. Further increasing the pH to 9.0 resulted in a reproducible significant kill rate of greater than 2 logs for both MMV and REO viruses. This process change is technically easy to implement and the exposure time of rVWF to the high pH environment can be kept relatively low (maximum 6 hours). Elution of bound rVWF is carried out under neutral conditions.

The analysis of the process capacity for virus inactivation was carried out according to the recommendations of the CPMP guideline 268/95, using the following formula:

where

R = virus reduction factor

Vi = volume of starting material [ml]

Ti = concentration of virus in the starting material [TCID50/ml]

V2 = volume of material after the step [ml]

T2 = virus concentration after passage [TCID50/ml]

The volumes and titers of each virus-added sample before and after treatment were used to calculate R. As long as the virus was undetectable, the detection limit was taken as the virus titer for calculation. Calculations were carried out with various titers (logiü[TCID50/mL] given to two decimal places, and only final results, i.e. reduction factors (R) were rounded to one decimal place.

Example 2

The UNO S eluate was concentrated to approximately 800 µg rVWF antigen/ml by ultrafiltration using 30 kDa cutoff modified cellulose membranes to facilitate analysis of trace impurities and product variants.

rVWF analysis

Ristocetin activity. Ristocetin Cofactor Activity is determined by a turbidimetric analyzer using a von Willebrand reagent containing stabilized thrombocytes and the antibiotic "ristocetin". The von Willebrand Factor contained in the sample (= Ristocetin Cofactor) causes agglutination of stabilized thrombocytes in the presence of ristocetin. Agglutination reduces the turbidity of the reagent preparation, and the change in optical density is measured by the turbidimetric analyzer. Calibration is carried out using the WHO concentrate reference standard No. 00/514.

VWF antigen. VWF samples were analyzed for their VWF antigen content in a double sandwich ELISA assay with two polyclonal antibodies. The measurement of the color reactions on the microtitre plates is carried out with a photometer at 490 nm. The concentration of each sample is calculated with respect to the standard curve with a computer assisted ELISA Analysis Program (curve algorithm: cubic regression). All readings are corrected against the blank test.

FVIII binding activity. The FVIII binding of rVWF under static conditions was determined by a chromogenic ELISA assay (ACE) incubating a constant amount of rFVIII with a sample containing diluted VWF. The formed VWF-FVIII complex was then transferred to a microtiter plate coated with a commercial rabbit anti-human VWF polyclonal antibody. After incubation, unbound FVIII was removed in a subsequent wash step. Bound FVIII was quantified by a commercial FVIII chromogenic assay (Technochrom FVIII: C reagent kit, Technoclone, Austria). Optical densities corrected against blank (in mOD/min at 405 nm) were plotted as a function of VWF:Ag concentrations on a logarithmic scale.

SDS-PAGE analysis. A standard 8% SDS-PAGE analysis under reduced conditions and staining of the gels with Coomassie blue and silver staining can provide an idea of the protein composition of rVWF. After transferring the separated protein bands to a nitrocellulose membrane and immunostaining the protein with appropriate antibodies against VWF, FVIII, and Furin, respectively, a comparison of the VWF-related proteins and the proteins can be made. totals.

Multimeric analysis. The multimeric structure of VWF is analyzed by high density horizontal SDS agarose gel electrophoresis. Briefly, samples are diluted to the same concentration in the range 0.3-1.0 IU/mL VWF:Ag and incubated with Tris-EDTA-SDS buffer, and multimers are separated under non-reducing conditions. on an agarose gel. VWF multimers were visualized by gel immunostaining with rabbit anti-human VWF polyclonal antibody, followed by alkaline phosphatase (ALP)-conjugated goat anti-rabbit IgG using the ALP Color Developer Kit. Alternatively, agarose gels were transferred to a blotting membrane and staining by rabbit anti-human VWF polyclonal antibody followed by horseradish peroxidase-conjugated anti-rabbit IgG. Electrochemiluminescence was used for visualization, which increases the sensitivity of VWF detection by at least two magnitudes. To analyze the size distribution of VWF multimers and the multimeric structure, low resolution (1% agarose) and high resolution (2.5% agarose) conditions were used, respectively.

HPLC analysis. recombinant VWF can be cleaved by GluC (V8 protease) under native conditions to obtain two main fragments (N-terminal and C-terminal homodimeric fragment), which are separated on a C4 HPLC column in reverse phase. The fragments are detected by monitoring the UV absorbance at 280 nm.

Peptide mapping. The primary structure of rVWF was investigated using a peptide mapping method. Purified rVWF samples were reduced with dithiothreitol (DTT) and free sulfhydryl groups were blocked with 4-vinylpyridine. Sequencing grade trypsin was then added to the rVWF and allowed to react for 18 hours. The resulting peptide mixture was separated by reverse phase chromatography. Eluting peptides were detected by online UV detection at 214 nm and online electrospray ionization mass spectrometry.

Deamidated rVWF analysis. The analytical method for the detection of isoaspartate (a reaction product from the deamidation of asparagine) employs tryptic digestion, followed by enzymatic Protein Isoaspartyl Methyltransferase (PIMT) reaction using the ISOQUANt IsoAspartate Detection Kit supplied by Promega. PIMT catalyzes the transfer of a methyl group from the substrate S-adenosyl-L-methionine (SAM) to IsoAsp at the carboxyl position, generating S-adenosyl-homocysteine (SAH). Stoichiometrically released SAH is detected at a wavelength of 260 nm by a RP HPLC method.

The analytical data of the product obtained through the different processes are summarized in Table 2. T l 2: D n lí in H nrl

As can be seen in Table 2, the biochemical properties of rVWF purified by the different process variants are comparable.

The main band of rVWF protein is very similar in all products, while the magnitude of impurities is less in sample #1 (designated as VWF #7 in Figure 1) by both silver staining and staining. Western blot analysis to determine residual rFVIII. The rFVIII banding pattern is comparable between all batches, suggesting that no degradation has occurred due to the pH 9.0 conditions.

Low and high resolution agarose gel electrophoresis revealed the high similarity of the rVWF preparations. Using low resolution multimeric analysis, no differences in multimeric composition could be observed. Figure 2 shows the stained gel of UNO S passages with samples with added MMV and REO virus. Furthermore, high-resolution multimeric analysis revealed the intact multimeric pattern, suggesting that no damage to the rVWF multimers occurred due to the pH 9.0 conditions. Figure 3 shows the stained gel of UNO S passages with samples with added MMV and REO virus.

No increase in deamidation could be detected by the Isoquant assay due to the residence time of rVWF at pH 9.0. In general, the mole percent of deamidated rVWF is very low. Subjecting the purified rVWF preparations obtained by the process variants to proteolytic digestion by native V8 protease (see Figure 4) or trypsin (see Figure 5) in the denatured state and separating the resulting peptides by RP -HPLC resulted in similar chromatograms for all samples.

The minor differences in the peak patterns are due to the presence of different amounts of impurities (mainly residual rVWF propeptide, as can be seen in Figure 1) in the preparations, which was confirmed by mass spectrometry or N-sequence analysis. -terminal.

Example 3

Purification of rVWF by high pH cation exchange chromatography with addition of MMV virus. A column packed UNOsphere S resin was activated with 1 CV of 2M NaCl and equilibrated with 25 CV of equilibration buffer (pH = 9.0). Then, a solution containing rVWF adjusted to a conductivity of 15 mS/cm and a pH of 9.0 and with addition of mouse minute virus (MMV) was loaded onto the column at a linear flow rate of approximately 10.0 cm/ h. The column was then washed with 10 CV of equilibration buffer (pH=9.0) and the product eluted with 3.5 CV of elution buffer (pH=7.5) at a linear flow rate of 65 cm/h. The increased pH during the loading and washing phase significantly reduced the binding of virus particles to the resin, but preserved full binding of product VWF. As a result, most of the loaded virus particles were in the unbound (continuous flow) fraction and Separate wash of product that was recovered in the common eluate load in high yields. The results in Table 3 show that by applying this procedure a virus clearance capacity of 2 logs could be achieved with the non-enveloped mouse minute virus (MMV) model.

The TCID50 assay was carried out as explained below. Briefly, 1/2 log serial dilutions of the samples were prepared in the appropriate tissue culture medium and 100 µl of each dilution was added to each of 8 wells of a microtiter plate seeded with the indicator cell line. The cells were incubated for 7 days at 36°C+2°C before evaluating the cytopathic effect by visual inspection of the cells under a microscope. Mean tissue culture infectious doses (TCID50) were calculated according to the Poisson distribution and expressed as logio[TCID50/ml].

Table 3: RVWF purification in UNOs here S

Purification was carried out using a column with a diameter of 15 mm and a bed height of 14 cm. Data shown are viral titers of active mouse minute virus.

Example 4

Purification of rVWF by high pH cation exchange chromatography with addition of Reo virus type 3.

A column packed UNOsphere S resin was activated with 1 CV of 2M NaCl and equilibrated with 25 CV of equilibration buffer (pH = 9.0). Then, a solution containing rVWF adjusted to a conductivity of 15 mS/cm and a pH of 9.0 and with addition of various non-enveloped viruses was loaded onto the column at a linear flow rate of approximately 100 cm/h. The column was then washed with 10 CV of equilibration buffer (pH=9.0) and the product eluted with 3.5 CV of elution buffer (pH=7.5) at a linear flow rate of 65 cm/h. The increased pH during the loading and washing phase significantly reduced the binding of virus particles to the resin, but preserved full binding of product VWF. As a result, most of the loaded virus particles were in the unbound (flow-through) and wash fraction separated from the product which was recovered in the common eluate load in high yields. The results in Table 4 show that by applying this procedure a virus clearance capacity of 2 logs could be achieved with the non-enveloped model of mouse Reo virus type 3 (REO-III).

Table 4: RVWF purification in UNOs here S

Purification was carried out using a column with a diameter of 15 mm and a bed height of 14 cm. Data shown are active Mouse Reovirus Type III (REO-III) viral titers.

Example 5

Purification of rVWF on UNOsphere S according to the standard procedure (neutral pH).

A column-packed UNOsphere S resin was activated with 1 CV of 2M NaCl and equilibrated with 25 CV of equilibration buffer (pH = 6.5). Then, a solution containing rVWF adjusted to a conductivity of 15 mS/cm and a pH of 6.5 and with addition of various non-enveloped viruses was loaded onto the column at a linear flow rate of approximately 100 cm/h. The column was then washed with 10 CV of equilibration buffer (pH = 6.5) and the product eluted with 3.5 CV of elution buffer (pH = 7.5) at a linear flow rate of 65 cm/h. The viral titer of the various viruses analyzed was evaluated in the different chromatographic fractions (loading, continuous flow column, wash, eluate, post-eluate) and the reduction factors were calculated. The results in Table 5 show that by applying the standard purification procedure for VWF on UNOsphere S, the non-enveloped virus killing capacity was insufficient for the different viruses. model analyzed to claim a strong chromatographic step for non-lipid enveloped virus removal.

Table 5: Purification of VWF according to the standard procedure and the capacities of r i n rr n i n r vir in nv l r

The reduction rate is calculated as the total viral load in the load fraction divided by the total viral load in the eluate fraction expressed in logarithmic values.

Claims (5)

1. Method for removing a virus without a lipid envelope from a solution containing protein, comprising: applying the solution to a cation exchange resin with a pH 3 or more pH units above the isoelectric point of the protein; and washing the cation exchange resin with a wash buffer to form an eluate, said wash buffer having a pH lower than that of the solution applied to the cation exchange resin, the protein in the solution being a polypeptide having a molecular mass of at least 150 kilodaltons, and where non-enveloped virus is removed from the protein-containing solution. The method according to claim 1, characterized in that the cation exchange resin has a negatively charged group selected from the group consisting of carboxymethyl (CM), sulfoalkyl (SP, SE), sulfated cellulose esters, heparin and methyl sulfonate (S ). 3. Method according to any of claims 1-2, characterized in that the protein is a blood coagulation protein. The method according to claim 3, characterized in that the blood coagulation protein is selected from the group consisting of factor VIII and von Willebrand factor. 5. Method for removing virus without lipid envelope from a solution containing von Willebrand factor (VWF), comprising: applying the solution to a cation exchange resin with a pH 3 units or more above the isoelectric point of VWF, and washing the cation exchange resin with a first wash buffer to form an eluate, said first wash buffer having a pH lower than that of the solution applied to the cation exchange resin.